A Novel D-Psicose 3-Epimerase from Halophilic, Anaerobic Iocasia fonsfrigidae and Its Application in Coconut Water.

D-Psicose is a rare, low-calorie sugar that is found in limited quantities in national products. Recently, D-psicose has gained considerable attention due to its potential applications in the food, nutraceutical, and pharmaceutical industries. In this study, a novel D-psicose 3-epimerase (a group of ketose 3-epimerase) from an extremely halophilic, anaerobic bacterium, Iocasia fonsfrigidae strain SP3-1 (IfDPEase), was cloned, expressed in Escherichia coli, and characterized. Unlike other ketose 3-epimerase members, IfDPEase shows reversible epimerization only for D-fructose and D-psicose at the C-3 position but not for D-tagatose, most likely because the Gly218 and Cys6 at the substrate-binding subsites of IfDPEase, which are involved in interactions at the O-1 and O-6 positions of D-fructose, respectively, differ from those of other 3-epimerases. Under optimum conditions (5 µM IfDPEase, 1 mM Mn2+, 50 °C, and pH 7.5), 36.1% of D-psicose was obtained from 10 mg/mL D-fructose. The IfDPEase is highly active against D-fructose under NaCl concentrations of up to 500 mM, possibly due to the excessive negative charges of acidic amino acid residues (aspartic and glutamic acids), which are localized on the surface of the halophilic enzyme. These negative charges may protect the enzyme from Na+ ions from the environment and result in the lowest pI value compared to those of other 3-epimerase members. Moreover, without adjusting any ingredients, IfDPEase could improve coconut water quality by converting D-fructose into D-psicose with a yield of 26.8%. Therefore, IfDPEase is an attractive alternative to enhancing the quality of fructose-containing foods.

Characterization of 3-isopropylmalate dehydrogenase from extremely halophilic archaeon Haloarcula japonica.

3-Isopropylmalate dehydrogenase (IPMDH) catalyzes oxidative decarboxylation of (2R, 3S)-3-isopropylmalate to 2-oxoisocaproate in leucine biosynthesis. In this study, recombinant IPMDH (HjIPMDH) from an extremely halophilic archaeon, Haloarcula japonica TR-1, was characterized. Activity of HjIPMDH increased as KCl concentration increased, and the maximum activity was observed at 3.0 m KCl. Analytical ultracentrifugation revealed that HjIPMDH formed a homotetramer at high KCl concentrations, and it dissociated to a monomer at low KCl concentrations. Additionally, HjIPMDH was thermally stabilized by higher KCl concentrations. This is the first report on haloarchaeal IPMDH.

Expression and functional study of VpV262 Pol, a moderately halophilic DNA polymerase from the Vibrio parahaemolyticus phage VpV262.

Halophilic organisms are found widely in environments where the salt concentration is higher than 0.2 M. Halophilic proteins isolated from these organisms maintain structural integrity and function under high salt stress, whereas their non-halophilic homologs tend to aggregate and collapse. Here we report for the first time the expression and function of a DNA polymerase (DNAPol) VpV262 Pol, which belongs to DNAPol Family A from Vibrio parahaemolyticus phage VpV262. Enzymatic activity assay revealed that VpV262 Pol possessed 5'-3' polymerase activity as well as 3'-5' proofreading exonuclease activity. VpV262 Pol requires Mg2+ or Mn2+ to catalyze the polymerization reaction. Polymerization activity assay under a wide range of salt concentrations showed that VpV262 Pol maintains the highest polymerase activity with 0-0.3 M of NaCl/KCl and 0-0.5 M KAc (potassium acetate) /KGlc (potassium gluconate) when treated with 0-1 M corresponding salts, in contrast to significantly decreased activity of Phi29 Pol and Taq Pol above 0.2 M. Consistent with typical features of other halophilic proteins, negatively-charged amino acids are more frequently distributed on the surface of VpV262 Pol, contributing to highly solubility and enhanced halotolerance. While 3D-Structure of VpV262 Pol needs to be confirmed by experimental data further, this study here has added a member for the relatively small family of halotolerant DNA polymerase, and provides a valuable reference in isolation and characterization of DNA polymerases from halophilic organisms.

Site-saturation mutagenesis at amino acid 329 of Klebsiella pneumoniae halophilic α-amylase affects enzymatic properties.

Halophilic α-amylases possess optimal activity in high salt concentrations. Therefore, they can be used in many extreme conditions in industrialised production. In the present work, a halophilic α-amylase (KP) from Klebsiella pneumoniae was characterised, and it exhibited a high specific activity of 3512 U/mg under optimal conditions of 2 M NaCl at 50°C and pH 6.5, but only 97 U/mg in the absence of salt. Furthermore, threonine at position 329 (Thr-329) was found to be related to the non-halophilic properties of KP according to PCR-based site-saturation mutagenesis. The activity of a mutant KP in which this threonine was replaced by aspartic acid was improved 14.6-fold compared with the native enzyme under salt-free conditions, and was increased by 14.8% in the absence of salt. Additionally, the optimal enzymatic properties of KP, including pH and temperature, were altered very little by the amino acid replacement. A further three halophilic α-amylases displayed similar mutational results. The findings provide a reference for bidirectional transformation of KP and similar halophilic enzymes.

Properties and Applications of Extremozymes from Deep-Sea Extremophilic Microorganisms: A Mini Review.

The deep sea, which is defined as sea water below a depth of 1000 m, is one of the largest biomes on the Earth, and is recognised as an extreme environment due to its range of challenging physical parameters, such as pressure, salinity, temperature, chemicals and metals (such as hydrogen sulphide, copper and arsenic). For surviving in such extreme conditions, deep-sea extremophilic microorganisms employ a variety of adaptive strategies, such as the production of extremozymes, which exhibit outstanding thermal or cold adaptability, salt tolerance and/or pressure tolerance. Owing to their great stability, deep-sea extremozymes have numerous potential applications in a wide range of industries, such as the agricultural, food, chemical, pharmaceutical and biotechnological sectors. This enormous economic potential combined with recent advances in sampling and molecular and omics technologies has led to the emergence of research regarding deep-sea extremozymes and their primary applications in recent decades. In the present review, we introduced recent advances in research regarding deep-sea extremophiles and the enzymes they produce and discussed their potential industrial applications, with special emphasis on thermophilic, psychrophilic, halophilic and piezophilic enzymes.

Improving the thermal stability of azoreductase from Halomonas elongata by introducing a disulfide bond via site-directed mutagenesis.

Azoreductases mainly reduce azo dyes, the largest class of colorants, to colorless aromatic amines. AzoH, a new azoreductase from the halophilic bacterium, Halomonas elongata, has been recently cloned and expressed in Escherichia coli. The aim of this study was to improve thermal stability of this enzyme by introducing new disulfide bonds. Since X-ray crystallography was not available, homology modeling and molecular dynamics was used to construct the enzyme three-dimensional structure. Potential disulfide bonds for increasing thermal stability were found using DIScover online software. Appropriate mutations (L49C/D108C) to form a disulfide bond were introduced by the Quik-Change method. Mutant protein expressed in E. coli showed increased thermal stability at 50 °C (increased half-life from 12.6 Min in AzoH to 26.66 Min in a mutated enzyme). The mutated enzyme could also tolerate 5% (w/v) NaCl and retained 30% of original activity after 24 H incubation, whereas the wild-type enzyme was completely inactivated. According to circular dichroism studies, the secondary structure was not altered by this mutation; however, a blue shift in intrinsic florescent graph revealed changes in the tertiary structure. This is the first study to improve thermal stability and salt tolerance of a halophilic azoreductase.

Levansucrase from Halomonas smyrnensis AAD6T: first halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized.

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.

Bioluminescent and structural features of native folded Gaussia luciferase.

The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one-subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable part that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five SS bonds. Despite wide application of Gaussia luciferase in biomedical research, its biochemical properties are still insufficiently studied due to the general problem of obtaining the proper folded Cys-rich proteins in bacterial cells. Here we report the properties of the proper folded Gaussia luciferase produced in insect cells using baculovirus expression system. This high purity luciferase reveals the highest activity at 15-20 °C but retains only ~20% activity at 37 °C that may hamper its application for in vivo assays. The maximum of bioluminescent activity of GpLuc is found at NaCl concentrations in the range of 1.0-1.5 M and, furthermore, a high NaCl concentration enhances luciferase stability to thermal denaturation, i.e. Gaussia luciferase displays the features characteristic of halophilic enzymes. The studies on bioluminescence kinetics at different coelenterazine concentrations obviously show a positive cooperativity of Gaussia luciferase with coelenterazine (Hill coefficient - 1.8 ±â€¯0.2; K0.5-2.14 ±â€¯0.17 µM). We suggest this effect to be rather due to the so-called kinetic cooperativity conditioned by conformational changes in response to substrate binding than to the presence of two catalytic sites.

Rational Design of an Efficient Halotolerant Enzymatic System for In Vitro One-Pot Synthesis of Cytidine Diphosphate Choline.

Salt accumulation often impedes cytidine diphosphate choline (CDP-choline) in vitro biosynthetic process. In this work a halotolerant in vitro enzymatic system is developed to solve this problem. It applies a halotolerant choline-phosphate cytidylyltransferase (CCT) obtained from rational design instructed by a unique strategy, which refers to one of the features of naturally occurring halophilic enzymes. By increasing acidic residues on protein surface where is most variable with respect to amino acid in the sequence alignment with other CCT, the mutants are obtained. The mutants represent higher catalytic activities and IC50 values (inhibit activity by 50%) at high-salt concentrations. Furthermore, when the halotolerant CCT is applied to in vitro one-pot biosynthesis of CDP-choline, the maximum titer and productivity are 161 ± 3.5 mM and 6.2 ± 0.1 mM L-1 h-1 , respectively. When acetate concentration increases, it still keeps relatively high reaction rate and is 2.2-fold higher than process using wild-type CCT (3.87 mM L-1 h-1 comparing with 1.74 mM L-1 h-1 ). This halotolerant system has great potential for industrial use, and the rational design concept can be applied to modify other enzymes, addressing the salt accumulation problem in in vitro systems, and gives insight into resolving by-product inhibition during reaction.

Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500 mM NaCl at pH 6.0, fourfold by 250 mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000 mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.